Impartment of virus-resistance with the use of plant protein binding to plant virus transport protein

ABSTRACT

A method for conferring resistance to a plant virus to plants is provided. The method comprises the step of introducing into cells of the plant a polynucleotide encoding a protein capable of binding to a movement protein of the plant virus. In one embodiment, the protein encoded by the polynucleotide contains a sequence indicated by position 1 to 86 of SEQ ID NO. 2, or contains the sequence having one or several amino acid substitutions, deletions, and/or additions, and binds to the movement protein of the plant virus. In one embodiment, the polynucleotide contains a nucleotide sequence indicated by position 14 to 271 of SEQ ID NO. 1 or a nucleotide sequence hybridizable to that nucleotide sequence under stringent conditions.

TECHNICAL FIELD

The present invention relates to a method for conferring plant virusresistance to plants. More preferably, the present invention relates toa method for conferring virus resistance to plants by expressing aprotein capable of binding to a plant viral movement protein in plants.

BACKGROUND ART

Some molds and bacteria invade plants by secreting a cell wall-degradingenzyme or the like. In contrast, plant viruses, such as tobacco mosaicvirus (TMV) and tomato mosaic virus (ToMV) belonging to plant RNAviruses, do not encode the gene of a cell wall-degrading enzyme or thelike, and can go through cell walls only after entering through aphysical scar or being transmitted by insects or fungi.

A highly infective virus, for example, tobacco mosaic virus, exhibitsmassive multiplication activity, such as production of 10⁶ progeny perday. Such a level of multiplication does not always lead to disease.Initially infected cells are only a part of a whole plant, and mostcells are not invaded. In many cases, a cell on the surface of a leaf isfirst infected with a virus and the virus transfers into a mesophyllcell in which the virus is in turn multiplied. The multiplied virusesspread into the surrounding mesophyll cells. In this manner, virusesspread throughout plant tissue. Such a process of a plant virusspreading to neighboring cells is called cell-to-cell movement. Virusesmove to vascular bundle sheath cells, sieve parenchyma, and thencompanion cells, and subsequently start to move from tissue to tissuethrough sieve elements. This movement is called long-distance movement(Saibo-Kogaku [Cellular Engineering], Special Issue,Syokubutu-Saibo-Kogaku [Plant Cellular Engineering] series, Vol. 8,Shujyunsha, pp. 146-155, Chapter 3 “Uirusu-Teikosei-no-tame-no-Senryaku[Strategy for Virus Resistance]”, p. 2, Yuichiro Watanabe,“Syokubutsu-Uirusu-no-Saibokan-Iko [Cell-to-Cell Movement of PlantVirus]”).

Examples of resistance reactions of host plants against viral infectionare: (1) suppression of the amount of multiplication of a virus, orsubstantially no disease symptoms despite multiplication of a virus(tolerance); (2) multiplication of a virus only in a cell which thevirus has first entered, and prevention of movement of the virus tosurrounding cells so that the virus cannot spread throughout a plant(subliminal infection); (3) multiplication of a virus in an infectedleaf, and suppression of long-distance movement from the infected leafto upper leaves, resulting in no systemic infection; and (4) rapidnecrosis of tissue at infection sites in an early stage of viralinfection, resulting in local necrotic lesion. Viruses are localizedwithin necrotic tissue or a surrounding portion thereof, therebyavoiding systemic infection (hypersensitive reaction). Based on thesefindings, efforts have been made to further elucidate resistancemechanisms. In the efforts, molecular research on virus resistance hasbeen conducted by physiological and biochemical analysis and geneticanalysis from view points of infecting viruses and host plants(Saibo-Kogaku [Cellular Engineering], Special Issue,Syokubutu-Saibo-Kogaku [Plant Cellular Engineering] series, Vol. 8,Shujyunsha, pp. 166-176, Chapter 3 “Uirusu-Teikosei-no-tame-no-Senryaku[Strategy for Virus Resistance]”, p. 3, Hideki Takahashi,“Uirusu-ni-tai-suru-Syukusyu-Teikosei [Host Resistance to Virus]”).

Plant viruses multiply via successive infection steps, such as genomereplication in individual cells, cell-to-cell movement throughplasmodesmata, and long-distance movement through sieve tubes(Carrington et al., (1996) Plant Cell 8, 1669-1681; Baker et al., (1997)Science 276, 726-733). In these stages, the viral movement isfacilitated by one or more virus-encoded proteins, called movementproteins (MPs) (Deom et al. (1992) Cell 69, 221-224). These shouldinteract with various host factors (Carrington et al., supra; Baker etal., supra). Functional domains have been characterized in the MPs of anumber of plant viruses. These include tobacco mosaic virus (TMV) andcucumber mosaic virus (CMV). In TMV, two RNA binding domains have beenidentified in the C terminal half of the MP (Citovsky et al., (1990)Cell 60, 637-647), while only one such domain was found in the Cterminal third of the CMV MP (3a protein; Vaquero et al., (1997) J. Gen.Virol. 78, 2095-2099). Taking advantage of such RNA binding ability,viruses are thought to move from cell to cell as nucleoprotein complexes(Lazarowitz and Beachy, (1999) Plant Cell 11, 535-548). MPs fromdifferent virus families form a tubular structure (van Lent et al.,(1991) J. Gen. Virol. 72, 2615-2623; Storms et al., (1995) Virology 214,485-493; Huang et al., (2000) Virology 271, 58-64); and increase thesize exclusion limit of plasmodesmata (Wolf et al., (1989) Science 246,377-379). MPs were found in association with various subcellularstructures, including endoplasmic reticulum, cytoskeleton, andplasmodesmata (Tomenius et al., (1987) Virology 160, 363-371; Atkins etal., (1991) J. Gen. Virol. 72, 209-211; Heinlein et al. (1995) Science270, 1983-1985; McLean et al., (1995) Plant Cell 7, 2101-2114; Reichelet al., (1999) Trends Plant Sci. 4, 458-463).

In recent years, host factors that interact with viral MPs have becomeof interest. In the tomato, Tm-2 and Tm-2² are reported as resistancegenes to tomato mosaic virus (ToMV) (Hall, (1980). Euphytica 29,189-197; Fraser (1990) Annu. Rev. Phytopathol. 28, 179-200. 25). Mutantvirus strains, overcoming the Tm-2 or Tm-2² phenotype, have amino acidsubstitutions in the MP. This suggests an interaction between MP and theresistance gene products (Meshi et al., (1989) Plant Cell 1, 515-522;Weber et al., (1993) J. Virol. 67, 6432-6438). Two homologous proteinsin the Nicotiana tabacum and Arabidopsis thaliana Dna J family wereidentified in a yeast two-hybrid screen to interact with tomato spottedwilt virus (TSWV) MP (Soellick et al., (2000) Proc. Natl. Acad. Sci. USA97, 2373-2378). Pectin methylesterase, localized at plasmodesmata, wasfound to interact with TMV MP. This suggests that this enzyme guides theMP and/or MP/RNA complex to plasmodesmata (Dorokhov et al., (1999) FEBSLett. 461, 223-228; Chen et al., (2000) EMBO J. 19, 913-920). The CMV 2bprotein, required for long-distance viral movement (Ding et al., (1995)EMBO J. 14, 5762-5772), was reported to interact with a tobacco proteinthat was very similar to a prokaryotic protein LytB, which is involvedin penicillin tolerance in bacteria (Ham et al., (1999) Mol. Cells9,548-555). Brigneti et al. (1998) EMBO J 17 6739-6746 proposed that CMV2b functions as a suppressor of posttranscriptional gene silencing inhost plants. Recently, Voinnet et al., (2000) Cell 103, 157-167 reportedthat potato virus XMP prevents the spread of the gene silencing signalin N. benthamiana.

Thus, there are a number of findings on plant viral MPs. Nevertheless,there has been no report relating to prevention of plant viral infectionand conferring virus resistance to plants.

DISCLOSURE OF THE INVENTION

The present inventors found that by causing a protein capable of bindingto a movement protein of a plant virus, which was identified by thepresent inventors, to be expressed in plants, the plant viral movementprotein is prevented from intracting with a protein of plant hosts forviral infection, thereby blocking cell-to-cell movement of the plantvirus and thus conferring resistance to the plant virus to the hostplants. Thus, the present invention was completed.

According to an aspect, the present invention relates to a method forconferring resistance to a plant virus to plants. The method comprisesthe step of introducing into cells of the plant a polynucleotideencoding a protein capable of binding to a movement protein of the plantvirus.

In one embodiment, the protein encoded by the polynucleotide contains asequence indicated by position 1 to 86 of SEQ ID NO. 2, or contains thesequence having one or several amino acid substitutions, deletions,and/or additions, and binds to the movement protein of the plant virus.

In another embodiment, the polynucleotide contains a nucleotide sequenceindicated by position 14 to 271 of SEQ ID NO. 1 or a nucleotide sequencehybridizable to said nucleotide sequence under stringent conditions.

In one embodiment, the plant virus is Tobamovirus, and preferably tomatomosaic virus (ToMV).

In one embodiment, the polynucleotide is derived from Brassicacampestris or Arabidopsis thaliana.

According to another aspect, a plant produced by the method of thepresent invention is also provided.

In one embodiment, the plant is a monocotyledonous plant or adicotyledonous plant. In one embodiment, the plant is tobacco.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows electrophoresis photographs showing binding of ToMV MP toMIP102. (A) E. coli having the pGEX-Bc2 plasmid was grown without IPTG(lane 1) or with IPTG. The total proteins extracted from the bacteriawere electrophoresed in 10% SDS-polyacrylamide gel and stained by CBB.(B) MP binding was assayed on a renatured protein blot. Proteinsresolved as (A) were assayed on PVDF membrane and probed with³²P-labeled GST-PKA-MP. (C) Affinity-purified GST-MIP102 (lane 1) andGST (lane 2) were electrophoresed in 10% gel and stained. (D) Proteinsresolved as (C) were blotted on PVDF membrane and assayed with³²P-labeled GST-PKA-MP as (B). Arrows show the positions of GST-MIP102and GST. Positions of molecular mass (kDa) markers are shown on theleft.

FIG. 2 is a diagram showing a nucleotide sequence of MIP102 cDNA and itsputative amino acid sequence. The underlined amino acid sequenceindicates a putative single-stranded DNA binding domain, which is usedfor alignment in FIG. 3. The unique HindIII and EcoRI sites used forSouthern blotting analysis are identified by double underlines. Arrows 1and 2 indicate the positions of introns revealed by genomic DNAanalysis.

FIG. 3 is a diagram showing multiple sequence alignment of MIP102 andAtKELP and similar sequences in the GenBank database. The upperalignment indicates comparison of a putative amino acid sequence ofMIP102 and KELP (AtKELP) of A. thaliana. ★ indicates matched amino acidresidues between MIP102 and AtKELP. The lower alignment indicatescomparison of various proteins in a putative single-stranded bindingdomain. MIP102, AtKELP and KIWI were aligned with homologous sequencesidentified in other organisms, such as human (PC4/p15) (Ge and Roeder,1994: Kretzschmar et al., (1994) Cell 78, 525-534), C. elegans (T13F2.2protein, GenBank gene number Z81122), and S. pombe (CAB10003.1protein,GenBank gene number Z97185-2).

FIG. 4 is an electrophoresis photograph showing genomic Southernhybridization of MIP102. B. campestris genomic DNA was digested withHindIII (lane 1), EcoRI (lane 2) or EcoRV (lane 3), fractioned through1.0% agarose gel, and transferred to a nylon membrane. This blot washybridized with ³²P-labeled 440-bp PstI-HindIII fragment of pGEX-Bc2(containing nucleotide 1 to 428 of the cDNA insert shown in FIG. 2).Positions of size (kb) markers are shown on the left.

FIG. 5 shows electrophoresis photographs showing binding of ToMV MP tothe N terminus of MIP102. (A) Affinity-purified GST-MIP102 (lane 1),GST-MIP102dN (lane 2), GST-MIP102dC (lane 3) and GST (lane 4) wereresolved in 10% SDS-polyacrylamide gel and blotted onto a PVDF membrane.After renaturation of the proteins, the blot was probed with ³²P-labeledPKA-MP. (B) Protein profile in the gel used in (A) was revealed by CBBstaining. Arrows show the positions of recombinant proteins. Brokenarrows indicate the occurrence of protein degradation. Positions ofmolecular mass (kDa) markers are shown on the left.

FIG. 6 shows photographs showing green fluorescence of plant cells dueto a reporter protein for viral cell-to-cell movement. (A) If the virusgenome moves to neighboring cells, a green fluorescence protein isproduced in the cells which in turn emit green light (the photographdesignated “Multiple Cells”). (B) In contrast, if the virus does notperform cell-to-cell movement, fluorescence is generated only in asingle cell (the photograph designated “One Cell”).

FIG. 7 is a graph showing the influence of co-introduction ofpART7-Bc2dC-HA and piL.G3on cell-to-cell movement. In FIG. 7, thevertical axis indicates the rate of cell-to-cell movement represented bythe fluorescence spot ratio (multiple cells/total number), while thehorizontal axis indicates the molar ratio of plasmids(pART7-Bc2dC-HA/piL.G3).

FIG. 8 is a graph showing the influence of co-introduction ofpART7-Bc2dC-HA and piL.erG3 (ER localized GFP expression plasmid) oncell-to-cell movement. In FIG. 8, the vertical axis indicates the rateof cell-to-cell movement represented by the fluorescence spot ratio(multiple cells/total number), while the horizontal axis indicates themolar ratio of plasmids (pART7-Bc2dC-HA/piL.erG3).

FIG. 9 is a construction diagram showing the expression plasmidpART27-Bc2-HA.a which produces an N-terminal binding region of MIP102.

FIG. 10 shows electrophoresis photographs showing expression of aprotein of interest in transformed tobacco. From the right in thefollowing order, (A) Coomassie Brilliant Blue (CBB)-stained gel, (B) theresult of use of HA tag antibodies (middle), and (C) the result of useof KELP antibodies. Of lanes in each result, the leftmost lane indicatesa molecular mass marker and remaining lanes indicate transformed tobaccostrains 101, 102, 105 and 118 from left in this order. The rightmostlane indicates a non-transformed tobacco (control).

FIG. 11 shows an electrophoresis photograph showing the result ofinvestigation of virus resistance due to ToMV particle infection. Theopposite end lanes indicate molecular mass markers, and the remaininglanes indicate transformed tobacco strains 101r, 102r, 105r and 118r(these were regenerates from transformed tobacco strains 101, 102, 105and 118), and a non-transformed tobacco (SR1) from left in this order.For each strain, the left lane indicates upper leaves (U) while theright lane indicates infected leaves (I). A band indicated by an arrowcorresponds to coat protein.

FIG. 12 shows electrophoresis photographs showing binding of ToMV MP toAtKELP. (A) Affinity-purified GST-AtKELP (lane 1), GST-MIP102 (lane 2),and GST (lane 3) were resolved in 10% SDS-polyacrylamide gel and blottedonto a PVDF membrane. After renaturation of the proteins, the blot wasprobed with ³²P-labeled PKA-MP. (B) Protein profile in the gel used in(A) was revealed by CBB staining. Arrows show the positions ofrecombinant proteins. The size of GST-MIP102 was larger than GST-AtKELPdue to extra 15 amino acids between GST and MIP102. Positions ofmolecular mass (kDa) markers are shown on the left.

FIG. 13 is an electrophoresis photograph showing binding of AtKELP toMPs of various viruses. Equal amount (1 μg) of affinity-purified GST-MP(ToMV), GST-CTMVMP (CTMV-W), GST-CMVMP (CMV), GST and bovine serumalbumin were immobilized onto a nitrocellulose membrane and probed with³²P-labeled PKA-AtKELP.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, a method for conferring plant virusresistance to plants is provided. By “conferring plant virus resistanceto plants” is intended that even if a plant is infected with a virus,disease damage is prevented or minimized. The method of the presentinvention comprises the step of introducing a polynucleotide encoding aprotein capable of binding to a plant viral movement protein into plantcells.

A protein encoded by a polynucleotide used in the method of the presentinvention binds to a movement protein (MP) of an infecting plant virus.This interaction blocks the interaction the infecting viral movementprotein and factors existing in a host plant, which may be involved inviral movement. Therefore, viral movement from cell to cell viaplasmodesmata may be blocked. When the above-described protein isexpressed in plants, plant viral cell-to-cell movement, and thus,tissue-to-tissue movement (long-distance movement) may be blocked. As aresult, viral disease damage is prevented or minimized. Therefore, thepolynucleotide used in the method of the present invention expresses aprotein capable of binding a plant viral movement protein in plants,thereby conferring plant virus resistance to the plants.

The polynucleotide used in the method of the present invention may bescreened for from a plant cDNA library as a polynucleotide encoding aprotein capable of binding to a movement protein (MP) encoded in a plantviral RNA genome. Hereinafter, a protein capable of binding to thismovement protein is referred to as a movement protein interactingprotein (MIP). Movement protein interacting protein (MIPs) may beidentified by a west western method which can search for an unknownprotein capable of binding to a known protein. For example, apolynucleotide encoding a protein capable of binding to a movementprotein (MP) encoded in the RNA genome of tomato mosaic virus (ToMV) maybe screened for from a cDNA library of a plant, such as Nicotianatabacum, Arabidopsis thaliana, and Brassica campestris. Such a MIPincludes, but is not limited to, for example, MIP204 derived from a N.tabacum library, and MIP102, MIP105 and MIP106 derived from a B.campestris library. Among these MIPs, MIP102 may exhibit the highestbinding to a tomato mosaic viral movement protein. The amino acidsequence of MIP102 and a nucleotide sequence encoding the same areindicated by SEQ ID NO. 2 and SEQ ID NO. 1.

The exemplary movement protein interacting protein MIP102 binds to amovement protein via the full length and a N terminal portion of MIP102.Therefore, in one embodiment, the polynucleotide of the method of thepresent invention encodes a protein containing an amino acid sequencefrom methionine (Met) at position 1 to glycine (Gly) at position 86 ofSEQ ID NO. 2 in the sequence listing. In another embodiment, thepolynucleotide used in the method of the present invention encodes aprotein containing an amino acid sequence from methionine (Met) atposition 1 to valine (Val) at position 165 of SEQ ID NO. 2 in thesequence listing. The polynucleotide used in the method of the presentinvention encodes a protein containing an amino acid sequence having oneor more amino acid deletions, substitutions, and/or additions as long asthe protein encoded by the polynucleotide has a function of binding to aviral movement protein.

In one embodiment, the polynucleotide of the present invention includesa polynucleotide having a nucleotide sequence of position 14 to 271 ofSEQ ID NO. 1 in the sequence listing. In one embodiment, thepolynucleotide of the present invention includes a polynucleotide havinga nucleotide sequence of position 14 to 508 of SEQ ID NO. 1 in thesequence listing.

Nucleotide sequences disclosed herein as well as fragments and variantsof proteins encoded by these sequences are encompassed by the presentinvention. By “fragment” is intended a portion of a nucleotide sequence,a portion of an amino acid sequence, and a protein encoded by thesequence. A fragment of a nucleotide sequence may encode a proteinfragment keeping one or more functional biological activities of anative protein. In the method of the present invention, a disclosednucleotide sequence and a fragment of a protein encoded by the sequencemay also be used as long as they bind to a viral movement protein.

A variant of a protein encoded by the polynucleotide of the presentinvention means a protein derived from a native protein by one or moreamino acid deletions (so-called shortening) or additions at the Nterminus and/or the C terminus of the protein; an amino acid deletion oraddition at one or more sites in the protein; or an amino acidsubstitution at one or more sites in the protein. Such a variant may begenerated by, for example, polymorphism or artificial manipulation.

A protein encoded by the polynucleotide of the present invention may bemodified by various methods (including amino acid substitution,deletion, shortening, and insertion). Methods for such manipulation aregenerally known in the art. For example, amino acid sequence variants ofa protein encoded by a plant gene capable of regulating stressresistance according to the present invention may be prepared bymutations in DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. For example, see Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra,editors, (1983) Techniques in Molecular Biology (MacMillian PublishingCompany, New York) and the references cited therein. Guidance as toappropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1987) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found. Washington, D.C., herein incorporated by reference).Conservative substitutions, such as exchanging one amino acid withanother having similar properties, may be preferred. Examples ofconservative substitutions include, but are not limited to substitutionsbetween hydrophobic amino acids (Ala, Ile, Leu, Met, Phe, Pro, Trp, Tyr,Val); hydrophilic amino acids (Arg, Asp, Asn, Cys, Glu, Gln, Gly, His,Lys, Ser, Thr); amino acids having aliphatic side chains (Gly, Ala, Val,Leu, Ile, Pro); amino acids having hydroxyl group containing side chains(Ser, Thr, Tyr); amino acids having sulfur atom containing side chains(Cys, Met); amino acids having carboxylic acid and amide containing sidechains (Asp, Asn, Glu, Gln); amino acids having base containing sidechains (Arg, Lys, His); amino acids having aromatic containing sidechains (His, Phe, Tyr, Trp).

Therefore, “have one or more deletions, substitutions and/or additions”means that a number of amino acids which may be substituted, deletedand/or added by polymorphism or artificial manipulation (including wellknown methods described above) may be substituted, deleted and/or added.“Have one or more deletions, substitutions and/or additions” also meansthat any number of amino acids may be deleted, added, and/or substitutedin the above-described amino acid sequence as long as the function of aprotein encoded by the polynucleotide of the present invention can beexpressed. It will be clearly appreciated by those skilled in the artthat influences of alterations, such as amino acid substitutions,deletions and/or additions, on activity depend on positions, degrees,types, and the like of amino acids to be altered. A protein encoded bythe polynucleotide of the present invention may have a number ofdeletions, substitutions and/or additions in the above-described aminoacid sequence, which number satisfies the following amino acid sequenceidentity, as long as the function of the protein of the polynucleotideof the present invention is expressed.

The polynucleotide used in the method of the present invention includesa polynucleotide having a nucleotide sequence encoding a protein havingan amino acid sequence, which has at least 70%, preferably at least 75%,more preferably at least 80%, even more preferably at least 90%, stilleven more preferably at least 95%, and most preferably at least 99%sequence identity with the amino acid sequence from Met at position 1 toGly at position 86 of SEQ ID NO. 2 in the sequence listing, such that aprotein encoded by this polynucleotide may bind to a viral movementprotein. The polynucleotide used in the method of the present inventionincludes a polynucleotide having a nucleotide sequence encoding aprotein having an amino acid sequence, which has at least 70%,preferably at least 75%, more preferably at least 80%, even morepreferably at least 90%, still even more preferably at least 95%, andmost preferably at least 99% sequence identity with the amino acidsequence from Met at position 1 to Val at position 165 of SEQ ID NO. 2in the sequence listing, such that a protein encoded by thispolynucleotide may bind to a viral movement protein.

The polynucleotide used in the method of the present invention includesa polynucleotide having a nucleotide sequence, which has at least 75%,preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, still even more preferably at least 95%, andmost preferably at least 99% sequence identity with a nucleotidesequence encoding the amino acid sequence from Met at position 1 to Glyat position 86 of SEQ ID NO. 2in the sequence listing (preferably, anucleotide sequence of A at position 14 to A at position 271 of SEQ IDNO. 1), such that a protein encoded by this polynucleotide may bind to aviral movement protein. The polynucleotide used in the method of thepresent invention includes a polynucleotide having a nucleotidesequence, which has at least 75%, preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, still evenmore preferably at least 95%, and most preferably at least 99% sequenceidentity with a nucleotide sequence encoding the amino acid sequencefrom Met at position 1 to Gly at position 86 of SEQ ID NO. 2 in thesequence listing (preferably, a nucleotide sequence of A at position 14to C at position 508 of SEQ ID NO. 1), such that a protein encoded bythis polynucleotide may bind to a viral movement protein.

The polynucleotide of the present invention may contain an additionalnucleotide sequence (e.g., a non-translated region) outside (i.e., atthe 5′ or 3′ terminus of) a nucleotide sequence encoding a proteincontaining the amino acid sequence of the above-described region.Preferably, the polynucleotide used in the method of the presentinvention consists of a full length sequence of position 1 to 913 of SEQID NO. 1. The polynucleotide of the present invention includes alldegenerate isomer of SEQ ID NO. 1. As used herein, the term “degenerateisomer” refers to DNA capable of encoding the same polypeptide whereonly degenerate codon(s) are different. For example, a DNA is called adegenerate isomer where a codon corresponding to an amino acid (e.g.,codon AAC corresponding to Asn) with respect to the DNA having a basesequence of SEQ ID NO. 1 is replaced with its degenerate codon (e.g.,codon AAT).

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or polynucleotide sequence, or the complete cDNA orpolynucleotide sequence.

As used herein, “comparison window” means includes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length, and optionally can be30, 40, 50, 100, or longer. Those skilled in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the polynucleotide sequence, a gap penalty is typically introducedand is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. A preferred method for determining the best overall match between areference sequence (the sequence of the present invention) and a subjectsequence is a homology analysis utilizing BLAST (Altshul et al., 1997,Nucleic Acids Res., 25, 3389-3402). In sequence alignment, the referencesequence and the subject sequence are both DNA sequences. An RNAsequence may be compared by converting U's to T's. The result of saidglobal sequence alignment is in percent identity. To calculate percentidentity, DNA sequence alignment may be conducted using the defaultparameters of BLAST.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins, it is recognizedthat residue positions which are not identical often differ byconservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those skilled in the art. Typically,this involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated as implemented inthe program PC/GENE (Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 80% sequenceidentity, preferably at least 85%, more preferably at least 90% and mostpreferably at least 95%, compared to a reference sequence using one ofthe alignment programs described using standard parameters. Thoseskilled in the art will recognize that these values can be appropriatelyadjusted to determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning and the like. Substantial identityof amino acid sequences for these purposes normally means sequenceidentity of at least 70%, more preferably at least 75%, 80%, 90%, andmost preferably at least 95%.

The terms “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70% sequence identityto a reference sequence, preferably 80%, more preferably 85%, mostpreferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman et al., J. Mol. Biol. 48:443 (1970). Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. Peptides which are“substantially similar” share sequences as noted above except thatresidue positions which are not identical may differ by conservativeamino acid changes. For peptide identity comparison, the GENETYX programmay be used. In this case, the default parameters of the program may beused.

A polynucleotide fragment encoding a biologically active portion of aprotein encoded by the polynucleotide used in the method of the presentinvention encodes at least 15, 25, 30, 50, 100, 125, 150, 175, 200 or225 contiguous amino acids, or the total number of amino acids of thefull length protein used in the method of the present invention (e.g.,243 amino acids in SEQ ID NO. 2). Generally, a fragment of a nucleotidesequence capable of conferring plant virus resistance to plants, whichis used as a hybridization probe for a PCR primer does not necessarilyencode a biologically active portion of a protein expressed by apolynucleotide capable of conferring plant virus resistance.

The exemplary movement protein interacting protein MIP102 is derivedfrom B. campestris. The polynucleotide used in the method of the presentinvention may also include polynucleotides encoding movement proteininteracting proteins derived from plants other than B. campestris. Sucha polynucleotide may be isolated by, for example, conducting PCR using aprimer designed based on the entirety or part of a disclosed nucleotidesequence and genomic DNA of a selected plant as a template, andthereafter, using the resultant amplified DNA fragment as a probe,screening a genomic DNA or cDNA library of the same plant. In thismanner, methods such as PCR, hybridization, and the like may be used toidentify such sequences based on the sequence identity to the sequenceset forth herein. Sequences isolated based on their sequence identity tothe entirety of the sequences set forth herein or fragments thereof, areencompassed by the present invention.

In a hybridization method, all or part of a known nucleotide sequencecan be used as probes which hybridize selectively to other correspondingnucleotide sequences existing in a group of cloned genomic DNA fragmentsor cDNA fragments derived from a selected organism (i.e., a genomelibrary or a cDNA library). These hybridization probes may be genomicDNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides,and may be labeled with a detectable group such as ³²P, or any otherdetectable marker. Preparation of probes for hybridization andconstruction of cDNA libraries and genomic libraries are generally knownin the art and is disclosed in Sambrook et al. (1989) Molecular Cloning:A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), herein incorporated by reference.

For example, the entirety or one or more portions of the nucleotidesequence of the plant gene capable of conferring plat virus resistanceset forth herein may be used as probes capable of hybridizingspecifically to the sequence of the plant gene capable of conferringvirus resistance to plants and messenger RNA thereof. In order toachieve specific hybridization under various conditions, such probes areunique between the sequences of plant genes capable of conferring virusresistance to plants, and preferably contain a sequence of at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes may be used so as to PCR amplify thesequences of plant genes capable of conferring virus resistance toplants from selected organisms. Methods for PCR amplification are wellknown in the art (PCR Technology: Principles and Applications for DNAAmplification, H A Erlich ed., Freeman Press, New York, N.Y. (1992); PCRProtocols: A Guide to Methods and Applications, Innis, Gelfland, Snisky,and White, eds., Academic Press, San Diego, Calif. (1990): Mattila etal. (1991) Nucleic Acids Res. 19:4967; Eckert, K. A. and Kunkel, T. A.(1991) PCR Methods and Applications 1:17: PCR, McPherson, Quirkes, andTaylor, IRL Press, Oxford, herein incorporated by reference). Thistechnique may be used as a diagnostic assay for isolating additionalcoding sequences from a desired organism or determining the presence ofcoding sequences in organisms. The hybridization technique includeshybridization screening of plated DNA libraries (either plaques orcolonies e. g., see Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2nd Ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.)).

Such sequence hybridization may be conducted under stringent conditions.The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than other sequences(e.g., at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe. Alternatively, stringency conditions can beadjusted to allow some mismatching in sequences so that lower degrees ofsimilarity are detected. Generally, a probe is less than about 1000nucleotides in length, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) and the temperature is atleast about 30° C. for short probes (pH 7.0 to 8.3) (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization at 42° C. in solutioncontaining 50% formamide, 4.4×SSC, 20 mM phosphate buffer (pH 6.8), 1×Denhardt's solution, 0.2% SDS and denaturated salmon sperm DNA (0.1mg/ml), a wash in 2×SSC containing 0.1% SDS at room temperature, and afinal wash in 0.2×SSC containing 0.1% SDS at 42° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The T_(m) is the temperature (under defined ionic strength andpH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desiredT_(m), those skilled in the art will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York): and Ausubel, et al.,Eds. (1995), Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). Also see Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.) (these are herein incorporatedby reference).

The base sequence of an obtained gene may be determined by a nucleotidesequence analysis method known in the art or a commercially availableautomatic sequencer.

The polynucleotide of the present invention may be typically obtained inaccordance with a method set forth herein or may be obtained by chemicalsynthesis based on the sequence disclosed herein. For example, thepolynucleotide of the present invention may be synthesized using apolynucleotide synthesizer (Applied BioSystems (at present, PerkinElmer), following the manufacturer's instructions.

A polypeptide produced in accordance with a procedure, such as a geneticengineering technique or a chemically synthesizing technique, asdescribed above may be confirmed to have desired activity, i.e.,conferring virus resistance as follows. An expression vector containingthe polynucleotide is produced. This expression vector is expressed inan appropriate cell to produce a protein. A procedure substantiallyidentical to that described in Example 1 below may be used to determinethat the protein binds to a plant viral movement protein (MP) ofinterest. Thereafter, an expression vector containing the polynucleotideis prepared. A procedure substantially identical to that described inExample 4 below may be used to confirm that when the protein isintroduced into plant cells together with viruses, the proteinsuppresses cell-to-cell movement of the viruses where a reporter gene,such as a green fluorescence gene, is used as an indicator.

The polynucleotide used in the method of the present invention confersplant virus resistance to plants. Plant viruses are not necessarilyspecific. A movement protein interacting protein capable of binding to amovement protein of a certain virus may bind to a movement protein ofanother virus. For example, MIP102 and AtKELP (a protein having about75% amino acid sequence identity to the full length of MIP102), whichare typical proteins capable of binding to a tomato mosaic viralmovement protein, also may bind to Brassicaceae mosaic virus (crucifertobamovirus) and cucumber mosaic virus.

In Examples set forth herein, virus resistance to tomato mosaic virus(ToMV) is shown for purposes of illustration. The method of the presentinvention may also confer resistance to other plant viruses to plants.Examples of plant viruses, for which resistance is sought, include, butare not limited to, viruses of the genera Tobamovirus, Tobravirus,Dianthovirus, Alfamovirus, Bromovirus, Cucumovirus, Comovirus,Nepovirus, Caulimovirus, Geminivirus, Potivirus, and Tospovirus. Thepolynucleotide used in the method of the present invention may beobtained by identifying a plant viral movement protein of a plant whichis to be conferred resistance, and screening for a protein capable ofbinding to the identified movement protein by a west western screeningmethod as described in Example 1.

The polynucleotide of the present invention may be linked to anappropriate plant expression vector using a method well known to thoseskilled in the art, and introducing the vector into a plant cell by aknown recombinant technique. The introduced gene is incorporated in DNAin the plant cell. Note that the DNA in the plant cell includeschromosomes as well as DNA contained in various organelles in the plantcell (e.g., mitochondrion, chloroplast, and the like).

As used herein, a “plant expression vector” refers to a nucleic acidsequence to which various regulatory elements, such as a promoter whichregulates expression of a gene of the present invention, are operativelylinked in host plant cells. The term “control sequence as used hereinrefers to a DNA sequence having a functional promoter and any relatedtranscription element (e.g., an enhancer, a CCAAT box, a TATA box, and aSPI site). The term “operatively linked” as used herein indicates that apolynucleotide is linked to a regulatory element which regulatesexpression of a gene, such as a promoter or an enhancer such that thegene can be expressed. Plant expression vectors may preferably includeplant gene promoters, terminators, drug-resistance genes, and enhancers.It is well known to those skilled in the art that the type of anexpression vector and the type of a regulatory element used may bechanged depending on the host cell. Plant expression vectors used in thepresent invention may have a T-DNA region. The T-DNA region can enhancethe efficiency of gene introduction, particularly when Agrobacterium isused to transform a plant.

The term “plant gene promoter” as used herein refers to a promoter whichis expressed in plants. A plant promoter fragment can be employed whichwill direct expression of the polynucleotide of the present invention inall tissues of regenerated plants. Examples of a promoter for structuralexpression include a promoter for nopaline synthase gene (Langridge,1985, Plant Cell Rep. 4, 355), a promoter for producing cauliflowermosaic virus 19S—RNA (Guilley, 1982, Cell 30, 763), a promoter forproducing cauliflower mosaic virus ³⁵S—RNA (Odell, 1985, Nature 313,810), a rice actin promoter (Zhang, 1991, Plant Cell 3,1155), a maizeubiquitin promoter (Cornejo 1993, Plant Mol. Biol. 23, 567), and theREXφ promoter (Mitsuhara, 1996, Plant Cell Physiol. 37, 49).

Alternatively, plant promoters can direct expression of thepolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are herein referred to as “inducible” promoters. Examplesof inducible promoters include promoters which are inducible byenvironmental conditions, such as infection or invasion of pathogens,injury of plants, light, low temperature, high temperature, dryness,ultraviolet irradiation, spray of a specific compound, or the like.Examples of such promoters include a promoter for a gene encodingribulose-1,5-diphosphate carboxylase small subunit which is induced bylight irradiation (Fluhr, 1986, Proc. Natl. Acad. Sci. USA 83, 2358), apromoter for the rice chitinase gene expressed due to infection orinvasion of molds, bacteria, or viruses (Xu, 1996, Plant Mol. Biol. 30,387), a promoter for the tobacco PR protein gene (Ohsima, 1990, PlantCell 2, 95), a promoter for the rice lip19 gene inducible by lowtemperature (Aguan, 1993, Mol. Gen. Genet. 240, 1), promoters for ricehsp72 and hsp8o genes inducible by high temperature (Van Breusegem,1994, Planta 193, 57), a promoter for the rab16 gene of Arabidopsisthaliana inducible by dryness (Nundy, 1990, Proc. Natl. Acad. Sci. USA87, 1406), and a promoter for the maize alcohol dehydrogenase geneinducible by ultraviolet irradiation (Schulze-Lefert, 1989, EMBO J. 8,651). A promoter for the rab16 gene is inducible by spraying abscisicacid which is a plant hormone.

A “terminator” is a sequence which is located downstream of a regionencoding a protein of a gene and which is involved in the termination oftranscription when DNA is transcribed into mRNA, and the addition of apoly A sequence. It is known that terminators contribute to thestability of mRNA, and have an influence on the level of geneexpression. Examples of such terminators include, but are not limitedto, a CaMV35S terminator and a terminator for the nopaline synthetasegene (Tnos).

A “drug-resistant gene” is desirably one that facilitates screening oftransformed plants. The neomycin phosphotransferase II (NPTII) gene forconferring kanamycin resistance, the hygromycin phosphotransferase genefor conferring hygromycin resistance, and the like may be preferablyused. The present invention is not so limited.

An enhancer” may be used so as to enhance the expression efficiency of agene of interest. As such an enhancer, an enhancer region containing anupstream sequence within the CaMV35S promoter is preferable. A pluralityof enhancers may be used for each plant expression vector.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′ terminus of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′terminus sequence to be added can be derived from, for example, thenopaline synthase or octopine synthase genes, or alternatively fromanother plant gene, or less preferably from any other eukaryotic gene.

Plant expression vectors as described above may be prepared using a generecombination technique well known to those skilled in the art. Inconstruction of a plant expression vector, pBI vectors, pUC vectors,pART vectors, or the like are preferably used. The present invention isnot so limited.

A plant material for DNA introduction can be appropriately selected fromleaves, stems, roots, tubers, protoplasts, calluses, pollen, embryos,shoot primordia, an the like according to the introduction method or thelike. A “plant cell” may be any plant cell. Examples of a “plant cell”include cells of tissues in plant organs, such as leaves and roots;calluses; and suspension culture cells. The plant cell may be in anyform of a culture cell, a culture tissue, a culture organ, or a plant.Preferably, the plant cell is a culture cell, a culture tissue, or aculture organ. More preferably, the plant cell is a culture cell.

A plant culture cell, to which DNA is introduced, is generally aprotoplast. DNA is introduced to a plant culture cell by aphysical/chemical method, such as an electroporation method, apolyethylene glycol method, or the like. A plant tissue, to which DNA isintroduced, is a leaf, a stem, a root, a tuber, a callus, pollen, anembryo, a shoot primordium, preferably a leaf, a stem, and a callus. DNAis introduced into a plant tissue by a biological method using avirus orAgrobacterium, or a physical/chemical method, such as a particle gunmethod, or the like. The method using Agrobacterium is disclosed, forexample, in Nagel et al. (Microbiol. Lett., 67, 325 (1990)). In thismethod, a plant expression vector is first used to transformAgrobacterium (e.g., by electroporation), and then the transformedAgrobacterium is introduced into a plant tissue by a well-known method,such as a leaf disc method. A particle gun method is described in, forexample, Klein et al. (1987), Nature 327:70-73; and Christon, P. PlantJ. (1992) 2,275-281. A protocol for transformation and a protocol forintroduction of a nucleotide sequence into plants vary depending on atarget plant to be transformed or the type of a plant cell (i.e.,monocotyledon or dicotyledon). Examples of an appropriate method forintroducing a nucleotide sequence into plant cells and subsequentlyinserting the sequence into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacteriummediated transformation (Townsend et al., U.S. Pat. No. 5,563,055),direct gene transfection (Paszkowski et al. (1984) EMBO J. 3:2717-2722),and ballistic particle acceleration (e.g., Sanford et al., U.S. Pat. No.4,945,050; Tomesra, U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No.5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment”, Plant Cell, Tissue, and Organ Culture: FundamentalMethods, Gamborg and Phillips ed., (Springer-Verlag, Berlin); and McCabeet al. (1988) Biotechnology 6:923-926). Also, see the followingreferences: Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477;Sanford et al. (1987) Particulate Science and Technology 5:27-37(onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soy bean);McCabe et al. (1988) Bio/Technology 6:923-926 (soy bean); Finer andMcMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh etal. (1998) Theor. Appl. Genet. 96:319-324 (soy bean); Datta et al.(1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl.Acad. Sci. USA85:4305-4309 (maize); Klein et al. (1988) Biotechnology6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S.Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) “Direct DNATransfer into Intact Plant Cells via Microprojectile Bombardment”, PlantCell, Tissue, and Organ Culture: Fundamental Methods, Gamborg ed.(Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol.91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize);Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bowenet al., U.S. Pat. No. 5,736,369 (crop species); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) The Experimental Manipulation of Ovule Tissues, Chapmann et al.eds. (Longman, New York) pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566 (whisker mediated transformation); D'Halluin et al.(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) PlantCell Reports 12:250-255, and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize (via Agrobacterium tumefaciens)) (all of these are hereinincorporated by reference). These methods are well known in the art. Amethod suitable for a plant to be transformed can be appropriatelyselected.

A cell, into which a plant expression vector has been introduced, isselected for drug resistance, such as kanamycin resistance. Thetransformed cell can be regenerated to a plant by a commonly usedmethod. See, for example, McCormick et al. (1986) Plant Cell Reports5:81-84.

A plant cell, into which the polynucleotide of the present invention hasbeen introduced, can be regenerated to a plant by culturing the plantcell in redifferentiation medium, hormone-free MS medium, or the like. Ayoung rooted plant can be grown to a plant by transferring it to soil,followed by cultivation. Redifferentiation methods vary depending on thetype of a plant cell. Redifferentiation methods for various plants aredescribed: rice (Fujimura, 1995, Plant Tissue Culture Lett. 2, 74);maize (Shillito, 1989, Bio/Technol. 7, 581; Gorden-Kamm, 1990, PlantCell 2, 603); potato (Visser, 1989, Theor. Appl. Genet. 78, 594); andtobacco (Nagata, 1971, Planta 99, 12).

Expression of an introduced gene of the present invention in aregenerated plant can be confirmed by a method well known to thoseskilled in the art. This confirmation may be carried out using, forexample, Northern blotting analysis. Specifically, total RNA isextracted from a plant leaf, is subjected to electrophoresis ondenaturing agarose, and is blotted to an appropriate membrane. This blotis subjected to hybridization with a labeled RNA probe complementary toa portion of the introduced gene, thereby detecting mRNA of a gene ofthe present invention. These plants may then be grown, and eitherpollinated with the same transformed strain or different strains, andthe resulting hybrid having the desired phenotypic characteristic may beidentified. Two or more generations may be grown to ensure that thesubject phenotypic characteristic is stably maintained and inherited andthen seeds harvested to ensure the desired phenotype or other propertyhas been achieved.

Plants which can be generated by the method of the present inventioninclude any plant to which a gene can be introduced. As used herein, theterm “plant” includes reference to whole plants, plant organs (e.g.,leaves, stems, roots, etc.), seeds, plant propagators (e.g., pollen),and plant cells, and progeny of same. Plant cells as used hereininclude, without limitation, seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, andmicrospores. The term “plant”includes monocotyledonous and dicotyledonous plants. Such plants includeany useful plants, particularly crop plants, vegetable plants, andflowering plants of garden varieties. The most preferable plant to whichthe present invention is applied is tobacco.

Examples of types of plants that can be used in the present inventioninclude plants in the families of Solanaceae, Poaceae, Brassicaceae,Rosaceae, Leguminosae, Cucurbitaceae, Lamiaceae, Liliaceae,Chenopodiaceae and Umbelliferae.

Examples of plants in the Solanaceae family include plants in theNicotiana, Solanum, Datura, Lycopersicon and Petunia genera. Specificexamples include tobacco, eggplant, potato, tomato, chili pepper, andpetunia.

Examples of plants in the Poaceae family include plants in the Oryza,Hordenum, Secale, Saccharum, Echinochloa and Zea genera. Specificexamples include rice, barley, rye, barnyard grass, sorghum, and maize.

Examples of plants in the Brassicaceae family include plants in theRaphanus, Brassica, Arabidopsis, Wasabia, and Capsella genera. Specificexamples include Japanese white radish, rapeseed, Arabidopsis thaliana,Japanese horseradish, and shepherd's purse.

Examples of plants in the Rosaceae family include plants in the Orunus,Malus, Pynus, Fragaria, and Rosa genera. Specific examples include plum,peach, apple, pear, Dutch strawberry, and rose.

Examples of plants in the Leguminosae family include plants in theGlycine, Vigna, Phaseolus, Pisum, Vicia, Arachis, Trifolium, Alfalfa,and Medicago genera. Specific examples include soybean, adzuki bean,kidney bean, pea, fava bean, peanut, clover, and bur clover.

Examples of plants in the Curcurbitaceae family include plants in theLuffa, Curcurbita, and Cucumis genera. Specific examples include gourd,pumpkin, cucumber, and melon.

Examples of plants in the Lamiaceae family include plants in theLavandula, Mentha, and Perilla genera. Specific examples includelavender, peppermint, and beefsteak plant.

Examples of plants in the Liliaceae family include plants in the Allium,Lilium, and Tulipa genera. Specific examples include onion, garlic,lily, and tulip.

Examples of plants in the Chenopodiaceae family include plants in theSpinacia genera. A specific example is spinach.

Examples of plants in the Umbelliferae family include plants in theAngelica, Daucus, Cryptotaenia, and Apitum genera. Specific examplesinclude Japanese udo, carrot, honewort, and celery.

Virus resistance conferred by the method of the present invention may beinherited to the primary generation of transformed plants as well assubsequent generations of the plants. The virus resistance conferred bythe peptide of the present invention may be exhibited in the primarygeneration of transformed plants and subsequent generations of theplants, propagators thereof (e.g., pollen), and seeds produced from thepropagators. Inheritance of the polynucleotide used in the method of thepresent invention into subsequent generations may be confirmed bysouthern analysis using a sequence of the polynucleotide disclosedherein as a probe.

The nomenclature used hereafter and the laboratory procedures describedhereafter often involve well known and commonly employed procedures inthe art. Standard techniques are used for recombinant methods,polynucleotide synthesis, cell culture, transformation and plantregeneration. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see, generally, Sambrook et al. Molecular Cloning: ALaboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference).

Hereinafter, the present invention will be described by way of examples.The present invention is not so limited. Materials, reagents, and thelike used in the examples are available from commercial sources, unlessotherwise mentioned.

EXAMPLES Example 1 Far-Western (“West Western”) Screening for MovementProtein Interacting Protein (MIP))

To identify plant proteins that bind to ToMV MP, the present inventorsconstructed N. tabacum and B. campestris expression cDNA libraries ofλGEX5. The construction of the cDNA libraries is the following. Leavesfrom N. tabacum cv. Samsun N N, stigma from B. campestris (S9/S9) andflower buds from A. thaliana ecotype Columbia were subjected to RNAisolation with QuickPrep Micro mRNA Purification Kit (Amersham PharmaciaBiotech), followed by cDNA synthesis with TimeSaver cDNA Synthesis Kit(Amersham Pharmacia Biotech). A λ phage vector λGEX5 (Fukunaga et al.,(1997) EMBO J. 16, 1921-1933; this vector has an IPTG inducible promoterand a cDNA-cloning site downstream of a GST reading frame) was used forconstruction of cDNA expression libraries. Phosphorylatedoligonucleotide adapters (5′-AGGTGCTGG-3′, 5′-CCAGCACCTGCA-3′) wereannealed and ligated to cDNAs to make compatible ends with the SfiI-cutvector. The cDNAs were ligated with the vector arms and subjected to invitro packaging. The phage libraries were amplified in E. coli strainBB4.

Far-western screening for MP interacting protein (MIP) was carried outbased on the protein-protein binding between each GST-fused cDNA productimmobilized on a membrane and GST-fused ToMV MP (GST-PKA-MP)phosphorylated with [γ-³²P]ATP for probing.

GST-fused ToMV MP (GST-PKA-MP) phosphorylated with [γ-³²P)ATP wasprepared as follows.

Initially, ToMV MP expression plasmid was prepared as follows. Theplasmid pGEX-30K for the expression of glutathione-S-transferase(GST)-fused ToMV MP (GST-MP) was described previously (Matsushita etal., (2000) J. Gen. Virol. 81, 2095-2102.). To add a consensusphosphorylation sequence for protein kinase A between GST and MP,synthetic oligonucleotides 30K-PKA1 (5′-AATTCGTCGTGCATCTGTTGC-3′) and30K-PKA2 (5′-AATTGCAACAGATGCACGACG-3′) coding for the five amino acidsRRASV were annealed and inserted into the EcoRI site of pGEX-30K in theproper orientation to generate pGEX-PKA-30K. This plasmid was used forproduction of a recombinant protein GST-PKA-MP. The 1.1-kb EcoRI-NotIinsert of pGEX-PKA-30K was placed between the EcoRI and NotI sites ofthe pGEX-6P-2 vector to construct pGEX-6P2-PKA-30K. This plasmid wasused for production of GST-P-PKA-MP, which could be cleaved byPreScission Protease (Amersham Pharmacia Biotech) to remove GST andprepare a recombinant protein PKA-MP.

Construction of pGEX-30KdA and pGEX-30 KdSX plasmids was describedpreviously (Matsushita et al., supra). The recombinant protein GST-MPDAencoded by pGEX-30 KdA had a deletion of the C-terminal 9 amino acidsthat were replaced by 27 artificial residues derived from the vector(QVALFGEMCAEPLFVYFSKYIQICIRS). Another recombinant protein, GST-MPdSX,encoded by pGEX-30 KdSX had a deletion of the C-terminal 31 amino acidsthat were replaced by 7 residues derived from the vector (LERPHRD).

Production and purification of recombinant proteins were conducted asfollows. Recombinant GST-fused proteins were produced in E. coliXL10-Gold (Stratagene) having appropriate plasmids and purified by usingGlutathione Sepharose beads (Amersham Pharmacia Biotech) as describedpreviously (Matsushita et al., supra). Anti-GST antibody (goat)purchased from Amersham Pharmacia Biotech was used to identify thefusion protein by western blotting analysis. Purified proteins werestored in NETN-D buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl,0.5% Nonidet P-40, 1 mM DTT).

³²P-labeled protein probes were prepared as follows. GlutathioneSepharose beads conjugated with about 1 μg of recombinant GST-fusedprotein were suspended in 200 μl of kinase buffer (50 mM Tris-HCl, 10mMMgCl₂, pH8.5) containing 3.7 MBq of [λ]-³²P]ATP (168 TBq/mmol) and 10units of the catalytic subunit of protein kinase A (New EnglandBioLabs). This reaction was continued for 30 min on a rotator at 30° C.and terminated by washing the beads 4 times with 1 ml of 50 mMTris-HCl(pH 8.0) buffer. The phosphorylated protein was eluted from beads with50 mM Tris-HCl (pH 8.0) buffer containing 10 mM glutathione. Whereindicated, the phosphorylated GST-fused protein was digested withPreScission Protease to remove GST domain before using as a probe forbinding experiments. The specific radioactivity of the probe was about1×10⁷ cpm/μg protein.

Far-western screening of the cDNA library was conducted as follows. E.coli strain BB was used for infection with the phage library. Thebacteria were incubated for 3 h at 42° C. to obtain plaques at a densityof 100 to 200/cm². The bacterial plates were overlaid withnitrocellulose filters wetted with 10 mM IPTG and further incubated for3.5 h at 37° C. The filters were washed and incubated in a blockingsolution, Block Ace (Dainippon-Pharm co.), for 1 h at 4° C. before theincubation with ³²P-labeled GST-PKA-MP (2×10⁵ cpm/ml) for 16 h at 4° C.The filters were then washed four times each for 5 min in PBS (137 mMNaCl, 2.68 mM KCl, 10.14 mM Na₂HPO₄, 1.76 mM KH₂PO₄, pH 7.4)supplemented with 0.2% Triton X-100 and subjected to autoradiography onBAS1500 system (Fuji Photo Film).

Several positive clones were isolated, such as MIP204 from the N.tabacum library, and MIP102, MIP105 and MIP106 from the B. campestrislibrary. Among these positive clones, MIP102 exhibited the highestbinding activity and was consequently selected for further analysis.

Phage DNA isolated from the MIP102 clone was used to reconstruct theexpression plasmid pGEX-Bc2 producing GST-fused MIP102, which was usedfor protein-binding assay (FIG. 1).

Protein-protein interaction was examined by a binding assay between³²P-labeled protein probe and target protein that was immobilized on amembrane. The target proteins were separated by SDS-polyacrylamide gelelectrophoresis and transferred onto PVDF membrane (Millipore). Afterwashing with buffer A (50 mM Tris-HCl, 20% 2-propanol, pH 8.0) andbuffer B (50 mM Tris-HCl, 5 mM β-mercaptoethanol, pH 8.0) each for 1 hat room temperature, the membrane was incubated for 1 h at roomtemperature in buffer B containing 6 M guanidine-HCl to denatureproteins. For renaturation, the membrane was rinsed for 5 min andincubated in buffer B containing 0.04% Tween 40 for overnight at 4° C.For a non-denatured system, proteins were directly immobilized on anitrocellulose membrane using a slot-blotter. The membrane was treatedwith Block Ace for 30 min at 4° C. and incubated with ³²P-labeledprotein probe in Block Ace supplemented with DNase I (18 μg/ml) andRNase A (60 μg/ml) for 4 h at 4° C. The filter membranes were washedfour times each for 5 min with PBS containing 0.2% Triton X-100 andsubjected to autoradiography analysis. The results are shown in FIG. 1.

On a Coomassie Brilliant Blue (CBB)-stained gel, a 48-kDa protein wasobserved when induced with IPTG (FIG. 1A, lane 2). Western blot analysisusing anti-GST antibody showed that this protein was GST-fused MIP102(data not shown). By using ³²P-labeled ToMV-MP probe, a specific bandwas detected at the position corresponding to the induced protein (FIG.1B, lane 2) while no such signal was observed in the un-induced lane(FIG. 1B, lane 1).

Subsequently, GST-MIP102 and GST were purified by using GlutathioneSepharose beads (FIG. 1C) and used for protein-binding assays with thesame probe (FIG. 1D). Purified GST-MIP102gave positive binding signal(FIG. 1D, lane 1) while purified GST did not (FIG. 1D, lane 2). Thisresult indicated that MIP102 clone was isolated by a specificinteraction with MP: it was not probed by GST-GST interaction.

Example 2 Analysis for cDNA and Genomic DNA of MIP102

The nucleotide sequence and its putative amino acid sequence of MIP102cDNA were determined as follows.

Phage DNA was prepared from the plate lysate of each clone by usingQIAGEN λ kit. For further analysis of the cDNA, phage DNA was digestedwith NotI to obtain the DNA fragment corresponding to pGEX-PUC-3T vectorwith cDNA insert. This DNA fragment was self-ligated to transform E.coli, from which the plasmid clone was recovered. The plasmid pGEX-Bc2recovered from the phage clone MIP102 was used to produce the GST-fusedprotein GST-MIP102.

The nucleotide sequences were determined by using the following primers:pGEX1 primer (5′ -GCAAGCCACGTTTGGTGGTG-3′), pGEX5 primer (5′-ATTTCCCCGAAAAGTGCCAC-3′), Bc2F03 (5′ -GAGCTTCCTTCTTCTAAAGG-3′), andBc2R02 (5′ -GCTTCGATAGCTGGAATATT-3′).

The nucleotide sequence of MIP102 cDNA (898 bp (excluding polyA)) isshown in FIG. 2. Homology searches using BLAST revealed that theputative protein was similar to a transcriptional coactivator KELP (75%identity) of A. thaliana (AtKELP; Cormack et al., (1998) Plant J. 14,685-692) (FIG. 3). For assessment of percentage of peptide identity,GENETYX program was used. MIP102 had 36% identity to KIWI of A. thaliana(Cormack et al., (1998) Plant J. 14, 685-692) (FIG. 3). Related proteinswere also found in several other taxonomically distinct organisms, suchas human (PC4/p15, 36% identity) (Ge and Roeder, (1994) Cell 78,513-523; Kretzschmar et al., supra) and S. pombe (protein having ProteinID CAB10003.1 in GenBank gene No. Z97185; 19% identity). Based on theN-terminal homology with AtKELP (FIG. 3), the first ATG codon wasassumed to be the initiation codon for MIP102. The open reading frame(ORF) encoded a 165 amino acid polypeptide with a calculated molecularmass of 19,227 Da and a pI to 4.8. A highly conserved region implicatedin single-stranded DNA-binding activity of PC4/p15 was identified(Kretzschmar et al., supra) (FIG. 3). The central region of MIP102 wasrich in glutamine and glutamic acid (amino acid position 66 to 81). Ahomopolymeric glutamine stretch was reported to increase transcriptionfactor potency (Gerber et al., (1994) Science 263, 808-811;Schwechheimer et al., (1998) Plant Mol. Biol. 36, 195-204).

To analyze the gene organization for MIP102, genomic DNA fragments wereamplified by PCR and their nucleotide sequences were determined. GenomicDNA was isolated from leaves of B. campestris by phenol/SDS method(Kingston, (1997) Phenol/SDS method for plant RNA preparation. In“Current Protocols In Molecular Biology” (V B Chanda Eds) Vol. 1, pp.4.3.1-4.3.3. John Wiley & Sons, Inc., New York). Genomic Southernhybridization was performed essentially as described by Sambrook et al.,(1989) “Molecular Cloning A Laboratory Manual” Cold Spring HarbourLaboratory Press, Cold Spring Harbor, N.Y. The DNA samples (10 μg) weredigested with appropriate restriction enzymes, fractioned through 1.0%agarose gel and transferred onto a positively charged nylon membrane(Millipore). The blot was hybridized with a ³²P-labeled probe for 16hours at 42° C. in a solution containing 50% formamide, 4.4×SSC, 20 mMsodium phosphate buffer (pH 6.8), 1× Denhardt's solution, 0.2% SDS anddenatured salmon sperm DNA (0.1 mg/ml). The membrane was washed severaltimes in 2×SSC containing 0.1% SDS at room temperature before the finalwashing for 30 min at 42° C. in 0.2×SSC containing 0.1% SDS.

Genomic DNA fragments corresponding to MIP102 cDNA were amplified by PCRusing two sets of primers: Bc2F04 (5′-GAAAACCCTAAAGATGGAG-3′) and Bc2R02(described above): Bc2F05 (5′-AATAAGCTTAACAGACGAAC-3′) and Bc2R07(5′-GATTTTAAAAGATCATTTTTGTCAT-3′). The PCR products were cloned into aTA cloning vector pCR2.1 (Invitrogen) and the nucleotide sequences weredetermined for three independent clones using the vector primers:Forward-ABI (5′-TGTAAAACGACGGCCAGT-3′) and Reverse-1(5′-GGAAACAGCTATGACCATG-3′) in addition to the internal primer Bc2F02and Bc2R02 described above.

Comparison of the nucleotide sequence of the genomic DNA with that ofcDNA revealed that the MIP102 gene contained two introns (FIG. 2) (73 bp(intron 1) and 278 bp (intron 2)). The locations of these introns werethe same as those for AtKELP (Cormack et al., supra). To estimate thenumber of genes encoding MIP102 homologues in B. campestris, a Southernblot hybridization using a 440 bp fragment of MIP102 cDNA was conducted(FIG. 4). The DNA probe hybridized with one band in each lane,indicating that MIP102 is encoded by a single copy gene in B.campestris.

Example 3 Binding Assay with Deletion Mutants of MIP102

GST-fused MIP102with N- or C-terminal deletions were produced and usedin a protein-binding assay as described in Example 1 to determine thedomain of MIP102 that is responsible for MP binding.

The plasmid pGEX-P-Bc2 was derived from the phage clone MIP102(described above). The pGEX-Bc2 BamHI-EcoRI fragment (842 bp) wasinserted between BamHI and EcoRI sites of pGEX-6P-2 (Amersham PharmaciaBiotech) to produce pGEX-P-Bc2.

In order to construct pGEX-P-Bc2dN, the DNA fragment for the C-terminalhalf of MIP102 was amplified by PCR from pGEX-Bc2 using Bc2FO2 primer(5′-AAYAARGARTTYGAYGAYGA-3′) and pGEX5 primer (described above), treatedwith T4 DNA polymerase and digested with NotI. The resultant 679-bpfragment was inserted between SmaI and NotI sites of pGEX-6P-2.

For the construction of pGEX-P-Bc2dC, the DNA fragment for theN-terminal half of MIP102 was amplified by PCR from pGEX-Bc2 using pGEX1primer (described above) and Bc2RO5 primer(5′-GAGACTCGAGTCATCCCTCCTTAGCTCTTT-3′), and digested with BamHI plusXhoI. The resultant 331-bp fragment was inserted between BamHI and XhoIsites of pGEX-6P-2.

pGEX-P-Bc2 was used for the expression of GST-fused MIP102 (GST-MIP102).pGEX-P-Bc2dN was used for GST-fused MIP102 (GST-MIP102dN) with deletionof N-terminal 86 amino acid residues while pGEX-P-Bc2dC was used forGST-fused MIP102 (GST-MIP102dC) with deletion of C-terminal 79 aminoacid residues. The coding sequences derived from PCR fragments wereconfirmed to have no errors.

As shown in FIG. 5A, ³²P-labeled PKA-MP bound to GST-MIP102dC containingthe N-terminal half (amino acid 1 to 86) of MIP102 (lane 3) as well asfull length GST-MIP102 (amino acid 1 to 165) (lane 1). The double bandsobserved in the lane of GST-MIP102dC (FIG. 5B, lane 3) may be due toprotein degradation near the C-terminus; the lower band with additionaldeletion showed the binding ability. In contrast, no MP-binding activitywas observed in GST (lane 4) and GST-MIP102dN containing the C-terminalhalf (amino acid position 87 to 165) of MIP102 (lane 2). This resultsuggests that the N-terminal half of MIP102 contains the MP bindingdomain.

Example 4 Inhibition of Cell-to-Cell Movement by the N Terminal Regionof MIP102

In order to allow for visual observation of ToMV movement, plasmidpiL.G3 was used in which the coat protein (CP) gene of the virus genomewas deleted and a green fluorescence protein (GFP) gene was insertedinto the virus genome in place of the CP gene. This plasmid was obtainedfrom Tetsuo Meshi of Kyoto University (Tamai and Meshi, Mol.Plant-Microbe Interact. (2001) 14, 126-134). Plasmid pART7-Bc2dC-HA wasalso prepared for expressing the C-terminal deletion MIP102(MIP102dC)obtained in Example 3. These preparation methods are described below.

In order to express MIP102dC, a plant expression binary vector pART7 wasused (Andrew P. Gleave, Plant Molecular Biology (1992) 20, 1203-1207).DNA fragments for the N-terminal half of MIP102 were amplified by PCRfrom pGEX-Bc2 using pGEX1 primer (described above) and Bc2R06HA primer(5′-TTGCTCTAGACTAAGCATAATCAGGAACATCATAAGGATATCCCTCCTTAGCTCTTTC-3′; thisBc2R06Ha primer contains an HA tag sequence) and digested with SmaI plusXbaI. The resultant about 350-bp fragment had a tag coding ahemaglutinin epitope (HA) fragment sequence inserted at the C terminusthereof. This fragment was inserted between SmaI and XbaI sitesdownstream of the CaMV35S promoter of pART7 to produce pART7-Bc2dC-HA.As a control plasmid, plasmid pART7-GUS which expresses GUS was alsoprepared.

Only plasmid piL.G3 or both of piL.G3 and pART7-Bc2dC-HA plasmids weresubjected to a particle gun method using leaf pieces of true leaves oftobacco (N. benthamiana) to introduce these plasmids into tobacco cells.This introduction of genes into cells by the particle gun method wasconducted using a BioRad gene introduction apparatus PDS-1000/He systemin accordance with the manufacturer's instructions. Green fluorescencewas observed under a fluorescence microscope. If the virus genome istransferred to neighboring cells, the green fluorescence protein isproduced in the cells containing the transferred virus genome so thatthe cells emit green light (FIG. 6, “Multiple Cells”). In contrast, ifthere is no cell-to-cell movement of the virus, fluorescence occurs inonly one cell (FIG. 6, “Single Cell”). Therefore, viral movement can bedetected according to a difference in fluorescence.

Next, a plasmid piL.G3 having GFP inserted therein instead of the CPgene and a plasmid which expresses the MP binding region of GUS(pART-GUS) or MIP102 (pART7-Bc2dC-HA) were subjected to a particle gunmethod using leaf pieces of true leaves of tobacco, while changing theratio of their amounts, to introduce these plasmids into tobacco cells.The result is shown in Table 1. TABLE 1 Transient Gene Introduction intoTrue Leaves of N. benthamiana by Particle Gun Number of FluoresenceSpots per Half Leaf^(a) Only one cell Multiple cells emits light emitslight Amount of DNA (portions without (portions with Multiple cells/Plasmid Combination per Introduction viral movement) viral movement)Total number^(b) piL. G3 + pART7-GUS 1.0 μg:2.5 μg 19 133  0.87 ± 0.094(ToMV/GFP) (control) piL. G3 + pART7-Bc2dC-HA 1.0 μg:2.5 μg 109 110 0.50± 0.11 (ToMV/GFP) (MP binding 0.5 μg:2.5 μg 47 46 0.49 ± 0.12 region ofMIP 102)^(a)The total number as a result of introduction into 3 to 4 leaves.^(b)Numbers following ± indicate standard deviations calculated from theresults of introduction into 3 to 4 leaves

The result shown in Table 1 revealed that expansion of greenfluorescence, which is a sign of virus genome movement, is inhibited bythe introduction of pART7-Bc2dC-HA. Therefore, it was considered that ifa protein at the N terminus of MIP102 is expressed in cells, thefunction of viral MP is suppressed so viral movement is inhibited.

The rate of cell-to-cell movement was observed while varying the molarratio of pART7-Bc2dC-HA and piL.G3. The result is shown in FIG. 7. InFIG. 7, the vertical axis indicates the rate of cell-to-cell movementrepresented by the fluorescence spot ratio (multiple cells/totalnumber), while the horizontal axis indicates the molar ratio of plasmids(pART7-Bc2dC-HA/piL.G3). As pART7-Bc2dC-HA was relatively increased,expansion of green fluorescence to multiple cells was decreased, i.e.,inhibition of movement was recognized.

Example 5 Effect of Co-Introduction of MIP102 Expression Plasmid When ERLocalized GFP Expression Plasmid was Used

The rate of cell-to-cell movement was observed while varying the molarratio of pART7-Bc2dC-HA and piL.erG3 (ER localized GFP expressionplasmid). piL. erG3 (ER localized GFP expression plasmid) was obtainedfrom Tetsuo Meshi of Kyoto University (Jun Tamai and TetsuoMeshi, TheProceedings of Annual Meeting of Japanese Society of Plant Pathology(2000), Okayama University).

The result is shown in FIG. 8. In FIG. 8, the vertical axis indicatesthe rate of cell-to-cell movement represented by the fluorescence spotratio (multiple cells/total number), while the horizontal axis indicatesthe molar ratio of plasmids (pART7-Bc2dC-HA/piL.erG3). As pART7-Bc2dC-HAwas relatively increased, expansion of green fluorescence to multiplecells was decreased, i.e., inhibition of movement was recognized.Comparing FIG. 8 with FIG. 7, it was revealed that the degree ofinhibition was greater when piL.erG3 (ER localized GFP expressionplasmid) was used than when ER non-localized GFP expression plasmid(piL.G3) was used.

Example 6 Construction of a Plasmid for Producing Transformed Plants byIntroduction of MIP Gene and Preparation of Transformed Plants Using thePlasmid

In order to produce transformed plants, phage DNA isolated from a MIP102clone was used to construct an expression plasmid pART27-Bc2dC-HA.awhich produces the N-terminal binding region of MIP102. A2.45-kb NotIfragment derived from pART7-Bc2dC-HA (described above) was inserted atthe NotI site of pART27 (obtained from Andrew P. Gleave, Plant MolecularBiology (1992) 20, 1203-1207) to produce pART27-Bc2dC-HA.a. Aconstruction diagram of this vector is shown in FIG. 9.

pART27-Bc2dC-HA.a was introduced into leaf pieces of tobacco by anAgrobacterium method. The Agrobacterium method was conducted inaccordance with Nagel et al. (Microbiol. Lett., 67, 325 (1990)). Theabove-described expression vector was used to transform Agrobacterium byelectroporation. Next, the transformed Agrobacterium was introduced intoplant tissue by a leaf disc method (Rabo-Manyuaru [Laboratory Manual],Syokubutsu-Idenshi-no-Kino-Kaiseki [Functional Analysis of Plant Gene]Masaki Iwabuchi and Toshiro Shimura, eds., 1992, Maruzen, pp. 31-56).Every two weeks, the above-described leaf pieces were subcultured andscreened for transformed tobacco cells based on the presence or absenceof kanamycin resistance due to expression of the kanamycin resistancegene derived from pART27 which was introduced into the tobacco cellsalong with the above-described polynucleotide. The selected transformedtobacco cells were redifferentiated to plants.

The transformed tobacco leaf having the introduced MIP102 was pulverizedin liquid nitrogen, followed by extraction in SDS-PAGE sample buffer at100° C. for 4 min. The extract was subjected to SDS-polyacrylamideelectrophoresis (SDS-PAGE, polyacrylamide gel concentration was 10 to20%), followed by western blotting analysis by a commonly used methodusing anti-KELP antibodies (produced by immunizing rabbits with purifiedKELP derived from pGEX-P-KELP (its production is described in Example 9below)) and anti-HA antibodies (obtained from MBL) as probes.

The result is shown in FIG. 10. (A) Coomassie Brilliant Blue(CBB)-stained gel, (B) the result of use of HA tag antibodies, and (C)the result of use of KELP antibodies. Of lanes in each result, theleftmost lane indicates a molecular mass marker and remaining lanesindicate transformed tobacco strains 101, 102, 105 and 118 from left inthis order. The rightmost lane indicates a non-transformed tobacco(control). In these results, a specific band of 14 kDa, which isconsidered to correspond to MIP102, was detected for the transformedtobacco strains 101, 103 and 118, but not 105 and the non-transformedtobacco (control).

Example 7 Investigation of Viral Cell-To-Cell Movement due toToMV-ΔCP/GFP Transcript Infection

In order to observe viral movement in the transformed tobacco having theintroduced MIP102, ToMV-ΔCP/GFP transcript product (RNA) was applied toleaves of the transformed tobacco strains having the introduced MIP102,101,103,105 and 118, their regenerated plants 101r, 103r, 105r and 118r,and the non-transformed plant (SR1). Note that regenerated plants wereproduced from leaf pieces in accordance with a commonly used method. Theapplication of the transcript was conducted as follows. pTL.G3 (obtainedfrom Tetsuo Meshi of Kyoto University; in this plasmid, the T7 promoteris transcribed upstream of the virus gene instead of the CaMV ³⁵Spromoter of piL.G3) was treated with T7 RNA polymerase to obtain RNAtranscripts. 1 μg of the RNA transcript was inoculated into tobaccoleaves having a width of about 3 to 5 cm. After two days, the infectedleaves were cut off and observed under a fluorescence microscope so asto observe fluorescence from GFP. The result is shown in Table 2. TABLE2 Infection of ToMV-ΔCP/GFP Transcript to Transformed TobaccoTransformed Number of Transformed Number of Tobacco Fluorescence SpotsTobacco Fluorescence Spots Individual Number One cell Multiple cellIndividual Number One cell Multiple cell 101 0 0 101r 0 0 103 0 0 103r 00 105 0 51 105r 0 3 118 0 0 118r 0 0 Non-transformant 1 0 0Non-transformant 2 0 6 101, 103 and 118 are MIP120dc high expressionplants Transformed Number of Number of Tobacco Fluorescence SpotsFluorescence Spots Individual Number One cell Multiple cell One cellMultiple cell 101r 0 1 0 4 103r 0 0 0 0 Non-transformant 1 0 48 0 45Non-transformant 2 0 108 0 108 101r, 103r, 105r, and 118r areregenerates from 101, 103, 105 and 118

As shown in Table 2, MIP102 was clearly expressed highly in Example 6 inthe transformants 101, 103 and 118, and their regenerates 101r, 103r and118r so that expansion of green fluorescence was suppressed in theseplants. In the plant 105 without a high level of expression, itsregenerate 105r, and the non-transformed plant (control), expansion ofgreen fluorescence was observed. Thus, it was revealed that viralmovement is inhibited in plants expressing MIP102.

Example 8 Investigation of Viral Infection by ToMV Particle Infection

To investigate whether resistance to ToMV infection was acquired by thetransformed tobacco having the introduced MIP102, viral particleinfection was conducted and then accumulation of viral coat protein (CP)in infected leaves and upper leaves was investigated. Leaves of theregenerates 101r, 103r, 105r and 118r of the transformed tobacco 101,103, 105 and 118 having the introduced MIP102, and the non-transformedplant (control) were infected with 0.06 μg of ToMV viral particles.After 10 days, the infected leaves and the leaves present at upperpositions (upper leaves) were cut off and pulverized in liquid nitrogen,followed by extraction in SDS-PAGE sample buffer at 100° C. for 4 min.The extract was subjected to SDS-polyacrylamide electrophoresis(SDS-PAGE, the polyacrylamide concentration was 10 to 20%) beforestaining the gel with Coomassie Brilliant Blue.

The result is shown in FIG. 11. The opposite end lanes indicatemolecular mass markers, and the remaining lanes indicate transformedtobacco strains 101r, 102r, 105r and 118r (these were regenerates fromtransformed tobacco strains 101, 102, 105 and 118), and anon-transformed tobacco (SR1) from left in this order. For each strain,the left lane indicates upper leaves (U) while the right lane indicatesinfected leaves (I). A band indicated by an arrow corresponds to coatprotein. This band was significantly observed in the infected leaves andupper leaves of the non-transformant SR1 and the transformant 105r whichdid not show a high level of expression of MIP102 in Example 6. Thetransformed strains 101r, 103r and 118r in which MIP102 was highlyexpressed and inhibition of viral movement was confirmed, accumulationof coat protein was reduced, so that virus resistance was expected.

Example 9 Binding Assay with AtKELP

To investigate whether ToMV MP binds to AtKELP having a high level ofhomology (75%) to MIP102, the cDNA for AtKELP was isolated from an A.thaliana cDNA library. A plasmid encoding GST-fused AtKELP (GST-AtKELP)was constructed and the purified fusion protein was used for aprotein-binding assay with ToMV MP as the probe. The protein-bindingassay was conducted as described in Example 3.

AtKELP expression plasmids were constructed as follows. The codingsequence of AtKELP (Cormack et al., supra) was amplified from A.thaliana 5′-Stretch cDNA library (Clontech) by PCR using KELP-F01 primer(5′-ACAGGAATTCCTAAAAATGGAGAAAGAGA-3′) and KELP-R01 primer(5′-TTGCCTCGAGTCAGACACGCGATTCCATTT-3′). The amplified 0.52-kb fragmentwas digested with EcoRI plus XhoI and inserted between EcoRI and XhoIsites of pGEX-6P-1 to produce pGEX-P-KELP. The coding sequence of KELPwas confirmed to have no base change. To add the phosphorylationsequence for protein kinase A, synthetic oligonucleotides KD-PKA1(5′-GATCACGTCGTGCATCTGTTG-3′) and KD-PKA2 (5′-GATCCAACAGATGCACGACGT-3′)were annealed and inserted into the BamHI site of pGEX-6P-1 to constructpGEX-6P1-3×PKA in which three sets of oligonucleotides were inserted inthe appropriate orientation downstream of the GST ORF. For theconstruction of pGEX-P-3×PKA-KELP, 0.52-kb EcoRI-XhoI fragment ofpGEX-P-KELP was cloned between EcoRI and XhoI sites of pGEX-6P1-3×PKA.pGEX-P-KELP and pGEX-P-3×PKA-KELP were used for expression of GST-AtKELPand GST-PKA-AtKELP, respectively.

As shown in FIG. 12, both GST-AtKELP (lane 1) and a positive controlGST-MIP102 (lane 2) showed MP-binding activity, while a negative controlGST did not show the activity.

Example 10 Binding Assay with MPs of CTMV-W and CMV

The present inventors also examined the binding ability of MIP102 andAtKELP to MPs of other plant viruses. In this case, the MPs of CTMV-Wand CMV were used. Protein binding assay was conducted as described inExample 3.

CTMV-W MP expression plasmids were constructed as follows. The plasmidpTW62 (Shimamoto et al., (1998) Arch. Virol. 143, 1801-1813) containingthe cDNA from a wasabi strain of crucifer tobamovirus (CTMV-W) wasdigested with SalI. The resultant 1.4-kb fragment containing the codingsequence of MP was inserted into SalI site of pAS2-1 (Clontech) in thesame orientation as that of the Gal4 gene. The resultant plasmid wasdigested with NcoI plus BamHI, treated with Klenow fragment andself-ligated to produce pAS-W30K in which the ORF of MP was continuousto that of Gal4. The 1.4-kb BamHI-NotI fragment of this plasmid wasinserted between BamHI and NotI sites of pGEX-5X-3 (Amersham PharmaciaBiotech) to produce pGEX-W30K. To adjust CTMV-W MP reading frame withthat of GST, pGEX-W30K was further digested with BamHI, treated withKlenow fragment and self-ligated to generate pGEX-W30KfB, which was usedfor expression of GST-fused CTMV-W MP (GST-CTMVMP).

CMV MP expression plasmids were constructed as follows. The plasmidpUCMVO3 (Hayakawa et al., (1989) J. Gen. Virol. 70, 499-504) containingthe RNA 3 cDNA derived from O strain of CMV (CMV-O) was digested withAvaI, treated with Klenow fragment and then digested with SalI toisolate 1.2-kb AvaI (blunted)-SalI fragment. This fragment containingthe 3a ORF (MP) was inserted between SmaI and XhoI sites of pGEX-6P-1 toconstruct pGEX-P-CMVOMP, which encodes GST-fused CMV MP (GST-CMVMP).

As shown in FIG. 13, ³²P-labeled PKA AtKELP bound to MPs of ToMV,CTMV-W, and CMV-O. When ³²P-labeled MIP102 was used as a probe, similarbinding activity was observed (data not shown).

The above-described examples illustratively describe various aspects ofthe present invention, how specific oligonucleotides of the presentinvention are produced, and how they are utilized. The present inventionis not so limited.

INDUSTRIAL APPLICABILITY

A polynucleotide capable of conferring virus resistance, which can beutilized in plant breeding and a method for conferring virus resistanceto plants using this polynucleotide are provided. Plants into which thepolynucleotide is introduced to confer resistance to plant virus arealso provided.

1. A method for conferring resistance to a plant virus to plants,comprising the step of introducing into cells of the plant apolynucleotide encoding a protein capable of being expressed in theplants and interacting with a movement protein of the plant virus.
 2. Amethod according to claim 1, wherein the protein encoded by thepolynucleotide contains a sequence indicated by position 1 to 86 of SEQID NO. 2, or contains the sequence having one or several amino acidsubstitutions, deletions, and/or additions, and binds to the movementprotein of the plant virus.
 3. A method according to claim 1, whereinthe polynucleotide contains a nucleotide sequence indicated by position14 to 271 of SEQ ID NO. 1 or a nucleotide sequence hybridizable to saidnucleotide sequence under stringent conditions.
 4. A method according toclaim 1, wherein the plant virus is Tobamovirus.
 5. A method accordingto claim 6, wherein the plant virus is tomato mosaic virus (ToMV).
 6. Amethod according to claim 1, wherein the polynucleotide is derived fromBrassica campestris or Arabidopsis thaliana.
 7. A plant, produced by themethod according to claim 1, monocotyledonous plant or a dicotyledonousplant.
 8. A plant according to claim 7, wherein the plant is amonocotyledonous plant or a dicotyledonous plant.
 9. A plant accordingto claim 8, wherein the plant is tobacco.